Two Types of Functionally Distinct NAD(P)H Dehydrogenases inSynechocystis sp. Strain PCC6803

Hiroshi Ohkawa,Himadri B Pakrasi,Teruo Ogawa

doi:10.1074/jbc.m003706200

Abstract

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome of Synechocystis sp. PCC6803 contains 6 ndhD genes. Three mutants were constructed by disrupting highly homologous ndhD genes in pairs. Only the DeltandhD1/DeltandhD2 (DeltandhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The DeltandhD3/DeltandhD4 (DeltandhD3/D4) mutant grew very slowly in air and did not take up CO(2). The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. TheDeltandhD5/DeltandhD6 (DeltandhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700(+) was high in DeltandhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity of Q(A) (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less in DeltandhD1/D2 and M55 than in DeltandhD3/D4 and DeltandhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1.NdhD2 type of NAD(P)H dehydrogenases.

Highlights

NdhD-less mutants, ⌬ndhD3 is the only mutant that displays the phenotype of slow growth at limiting CO2 (i.e. 50 ppm CO2) and reduced affinity for CO2 uptake, whereas the other ndhDless mutants (⌬ndhD1, ⌬ndhD2, ⌬ndhD4, and ⌬ndhD5) do not show such phenotype [6]
The ⌬ndhD1/⌬ndhD2 (⌬ndhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, the mutant grew normally under photoautotrophic conditions in air
It has been demonstrated that NDH-1 is essential for inorganic carbon (CO2 and HCO3Ϫ; designated Ci) transport in cyanobacteria [3,4,5,6,7,8,9,10,11], and it was assumed that ATP produced by NDH-1-dependent cyclic electron flow is essential to energize the Ci transport [7, 12]

Summary

EXPERIMENTAL PROCEDURES

The Type I NAD(P)H dehydrogenase complex (NDH-1) in cyanobacteria is involved in both the respiratory and photosynthetic electron transport chains [1]. The data base reveals that ndhD and ndhF are present as gene families with six and three members, respectively (note that NdhF4 has homology to NdhD5 and has been designated as NdhD6 [3]), most ndh genes are present as single copies This suggests that several types of NDH-1 exist in cyanobacteria, each with different NdhD and/or NdhF subunits, and with each potential complex having differing functions (4 – 6). Cells were suspended in 20 mM Tes-KOH (pH8.0) containing 15 mM NaCl, and the activities of respiration were measured as the rates of oxygen uptake 3 min after addition of 5 mM glucose, as described by Mi et al [12]. Wild-type and mutant cells used for P700 measurements were grown under 3% CO2 in air (v/v) to late logarithmic phase followed by incubation in darkness for 32 h. Wild-type and mutant cells were harvested in the logarithmic growth phase and suspended in fresh BG11 at a chlorophyll concentration of 2 ␮g mlϪ1. Pigments in the cells were extracted in methanol, and the concentration of chlorophyll in the extract was determined [20]

RESULTS

Oxygen uptake

DISCUSSION